Taxonomy, genetics and biogeography each make key contributions to biological conservation. However, integrating these disciplines to obtain a coherent account of the status of a taxon of concern is not always straightforward. This is the case for the cross-border endemic plant Physaria thamnophila (Brassicaceae). This US federally-listed endangered species is restricted to a set of unique geological sites just north of the Rio Grande (Rio Bravo del Norte) river in south Texas, USA. A single highly-disjunct occurrence of this species is found on a geologically and ecologically distinct site 260 km to the south, in Tamaulipas, Mexico. In this work, we quantify the genetic differentiation between the U.S. and Mexican populations using four microsatellite markers and sequences from three nuclear genes. In both sets of data, we find a high level of genetic divergence consistent with geographic isolation on a time scale of 1–2.5 million years. Further, we provide a hypothesis for the geological basis of this geographic isolation. Integrating our data with ecological, taxonomic and conservation considerations, we propose the sub-specific designation of Physaria thamnophila subsp. loretensis for the Mexican population. The evolutionary and conservation implications of this designation are presented. 

Plant collections and DNA sampling.—All Physaria species were formerly included in the genus Lesquerella (Al-Shebaz & O’Kane 2002). Tissues were obtained from 1) laboratory-germinated seedlings, 2) herbaria specimens as listed in Appendix 1. Additional tissue collections were made from P. thamnophila populations under permit and consultation with the US Fish and Wildlife Service. We sampled all known populations of P. thamnophila in which plants were present at the time of sampling. Four populations were in Zapata County, Texas, ten in Starr County, Texas, and one in Tamaulipast, Mexico. Approximately 0.5 cm² of tissue was used for each DNA isolation. DNAs were isolated from both fresh and dried tissue by a simple micro-scale preparation described previously (Pepper & Norwood, 2001). 

Microsatellite marker analyses.—Genomic DNAs from a single individual from the P. thamnophila SMR population (Starr County, Texas, U.S.A.) were used for microsatellite discovery. DNA fragments containing microsatellite (CT, CT and GG) repeats were captured using a biotinylated-oligonucleotide method described previously (Terry et al. 2006). All primers were designed to have a 45-60% GC content and a salt-adjusted (50 mM NaCl) melting temperature of 63–64°C. Amplifications were performed with Phusion Hi-Fi polymerase (New England Biolabs) using ±10 ng genomic DNA as a template. An annealing temperature of 58°C was employed for all reactions. PCR buffer and cycling conditions followed the manufacturer’s recommendations (New England Biolabs). Four microsatellite markers (Appendix 2) were amplified using fluorescently labeled primers (6-HEX and 6-FAM). Multiplexed (HEX + FAM) fragment analysis was performed using the ABI 3130 capillary DNA sequencer as described (Tarin et al. 2014). Fragment sizes were determined using GeneScan ver. 3.1 software (Applied Biosystems Inc.). Microsatellite genotyping was performed using Genotyper ver. 2.5 software (Applied Biosystems Inc.).  GeneAlEx ver. 6.5 (Peakall & Smouse 2012).

Microsoft Excel

GeneAlEx ver. 6.5 (Peakall & Smouse 2012), plug-in for Microsoft Excel.